Gao Fei, Hao Zhenzhen, Sun Xianhua, Qin Lina, Zhao Tong, Liu Weiquan, Luo Huiying, Yao Bin, Su Xiaoyun
1Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 South Zhongguancun Street, Beijing, 100081 People's Republic of China.
2College of Biological Sciences, China Agricultural University, Beijing, 100193 China.
Biotechnol Biofuels. 2018 Sep 24;11:261. doi: 10.1186/s13068-018-1264-z. eCollection 2018.
In the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. is a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive.
Herein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase promoter in , which allowed us to rapidly isolate cellulase hyperproducers from progenies transformed with a dedicated transcriptional activator and from an atmospheric and room temperature plasma-created mutant library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing and ) affecting regulatory stages beyond transcription. Using this method, cellulase hyperproducers were also successfully isolated from an -mediated random insertional mutant library.
The coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.
在生物燃料行业中，纤维素酶在将纤维素水解为可发酵葡萄糖的过程中发挥着不可或缺的作用。里氏木霉是一种常见的丝状真菌，具有突出的纤维素酶生产能力。虽然经典诱变和现代多重基因组工程都是提高纤维素酶产量的有效方法，但要成功获得具有提高的纤维素酶生产能力的菌株需要筛选大量菌株，这既耗时又费力。
在此，我们开发了一种通用方法，该方法将红色荧光蛋白（DsRed）在里氏木霉中的表达与萌发孢子的荧光辅助细胞分选（FACS）相结合。该方法首先通过在里氏木霉主要纤维素酶启动子的控制下在细胞内表达DsRed来建立，这使我们能够从用专用转录激活因子转化的里氏木霉后代以及从常压室温等离子体产生的突变体文库中快速分离出纤维素酶高产菌株。由于预期细胞内表达的DsRed主要用于分离影响纤维素酶转录的突变，因此通过将DsRed展示在里氏木霉细胞表面进一步改进了该方法，从而能够分离出具有影响转录以外调控阶段的有益基因改变（过表达和）的菌株。使用该方法，还成功地从里氏木霉介导的随机插入突变体文库中分离出了纤维素酶高产菌株。
DsRed-FACS偶联的高通量筛选方法被证明是快速分离里氏木霉纤维素酶高产菌株的有效策略，也可应用于其他具有工业重要性的丝状真菌。
